GE Healthcare

Life Sciences

Spectrophotometry
Handbook
Contents

Spectrophotometry basics 3
What is spectrophotometry? 3
Definition 3
Lambert’s Law 4
Beer’s Law 4

Nucleic acid applications 6

Direct UV measurement 7
A260 /A280 Ratio 7
A260 /A230 Ratio 7
A 320 Background correction 7
Nucleic acid measurements with low volume instruments 8

Protein applications 9
Protein concentration calculations 10
Christian and Warburg 10
Colorimetric methods 10
BCA 11
Biuret 11
Bradford 11
Lowry 11
2-D Quant Kit 11
Choosing your protein assay 12
Measuring proteins in low volume instruments 14

Other applications 15
Fluorescent dyes 15
Cell culture 15
Other molecules 16

Continued overleaf

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Spectrophotometry hints and tips 17
Choosing a cuvette 17
Beam heights 17
Choice of cuvettes 17
Buffer Compatibility 17
Low volume spectrophotometers 18
Choice of volume 18
Background correction 18

Which spectrophotometer? 19

Spectrophotometer selection guide 21

GE Healthcare spectrophotometer range 22
Datrys PC control software 22
Accessories overview 23

Glossary 24
Absorption 24
Bandwidth 24
Beam technology 25
Light source comparison 25
Methods of measurement 26
Monochromator 27
Pathlength 27
Stray Light 27
UV/Vis 27
Wavelength 27

References 28

Related products 29

2
Spectrophotometry basics
What is spectrophotometry?
Spectrophotometry is a scientific method based on the absorption of light by a substance,
and takes advantage of the two laws of light absorption.

Definition
spec·tro·pho·tom·e·ter/spektrōfōˈtämitər/

Noun: An apparatus for measuring intensity of light in a part of the spectrum,

as transmitted or emitted by particular substances.

Visible Light

Infrared Ultraviolet

Near Near

A B C

1 mm 3 µm 1400 nm 750 nm 700 nm 650 nm 600 nm 550 nm 500 nm 450 nm 400 nm 320 nm280 nm 200 nm

Fig 1. The electromagnetic spectrum. GE Healthcare Life Sciences offers a range of spectrophotometers which
operate in the UV, visible and near infrared section of the electromagnetic spectrum.

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Lambert’s Law (1)
The proportion of light absorbed by a medium is independent of the intensity of incident light.
A sample which absorbs 75% (25% transmittance) of the light will always absorb 75% of the
light, no matter the strength of the light source.

Lambert’s law is expressed as I/Io =T

Where I = Intensity of transmitted light

Io = Intensity of the incident light
T = Transmittance

This allows different spectrophotometers with different light sources to produce comparable
absorption readings independent of the power of the light source.

Beer’s Law (2)

The absorbance of light is directly proportional to both the concentration of the absorbing
medium and the thickness of the medium. In Spectrophotometry the thickness of the medium
is called the pathlength.

In normal cuvette-based instruments the pathlength is 10 mm. Beer’s law allows us to measure
samples of differing pathlength, and compare the results directly with each other.
GE Healthcare offers a variety of instruments and accessories which allow measurement of
pathlengths from 10 cm down to 0.2 mm.
1.5

1.2
Absorbance

0.9

0.6

0.3

0.0
0.0 0.2 0.4 0.6 0.8 1.0
Path Length (cm)

Fig 2. Beer’s Law: The absorbance of light is directly proportional to both the concentration of the absorbing medium
and the thickness of the medium.

4
Short pathlength instruments are used when the sample is of limited volume, scarce and maybe
requiring recovery, or is very concentrated (e.g. > 50 µg DNA/ml), and the user wishes to avoid
the need for dilution. Many samples would traditionally require dilution for two reasons:

1. So that there is enough volume to fill a 10 mm pathlength cuvette.

2. To lower the sample concentration enough to allow accurate measurement
by the spectrophotometer.

Dilution introduces an aspect of human error and can also prevent the use of that sample in
downstream applications.

To measure concentrated samples using a 10 mm pathlength would require a very powerful

light source to give transmittance that is high enough to be detected reliably. A shorter
pathlength reduces the absorbance – increasing the transmittance – hence reducing the
incident light required to achieve a reliable result. This removes the need to dilute the sample,
or to have a larger, more powerful or more expensive instrument.

When using short pathlengths (less than 10 mm), results are generally normalized to that
of a 10 mm pathlength, e.g. In the case of a 0.2 mm pathlength, the absorbance results are
multiplied by 50. However, at the same time any error from the system of absorption by the
cuvette is also multiplied by 50, increasing the possible effect on the result.

In basic terms: Absorbance = Concentration × Pathlength

Light
Source
Adjustable
Monochromator Aperture
Sample

Photoresistor
Amplifier
Spectrum Display

of light

Light is Light is
absorbed detected
Measure is
displayed
Fig 3. General schematic of a spectrophotometer.

5
Nucleic acid applications
Spectrophotometry can be used to estimate DNA or RNA concentration and to analyze the
purity of the preparation. Typical wavelengths for measurement are 260 nm and 280 nm.
In addition measurements at 230 nm and 320 nm can provide further information.

Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm. For pure samples
it is well documented that for a pathlength of 10 mm, an absorption of 1A unit is equal to a
concentration of 50 µg/ml DNA and 40 µg/ml for RNA. For oligonucleotides the concentration
is around 33 µg/ml but this may vary with length and base sequence.

So for DNA: Concentration (µg/ml) = Abs260 × 50.

These values are known as conversion factors.

A number of other substances which also absorb light at 260 nm could interfere with DNA values,
artificially increasing the result calculated from the absorption readings. To compensate for
this a selection of ratios and background corrections have been developed to help eliminate
false readings.

0.9 X: 300
Y: 0.063
0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1
×
0.0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)
Fig 4. A typical wavelength scan for a pure DNA sample.

6
There is a wide absorbance peak around 260 nm preceded by a ‘dip’ at 230 nm. Therefore
to measure the DNA absorption, the 260 nm DNA peak must be distinguishable from the
230 nm reading.

If the readings at 230 nm are too similar to those at 260 nm, DNA cannot be measured
accurately. Higher 230 nm readings can indicate contaminants in the sample. There should
also be a rapid tail-off from 260 nm down to 320 nm. For this reason, 320 nm is often used
to measure background (see background correction).

Direct UV measurement
A260/A280 Ratio
The most common purity check for DNA and RNA is the A260/A280 ratio. Any protein contamination
will have maximum absorption at 280 nm. Measurements are taken at both 260 nm and 280 nm
and compared to give a ratio. For DNA the result of dividing the 260 nm absorption by the
280 nm needs to be greater or equal to 1.8 to indicate a good level of purity in the sample.
For RNA samples this reading should be 2.0 or above. Results lower than this are indicative
of impurities in the sample.

A260/A230 Ratio
An increase in absorbance at 230 nm can also indicate contamination, which may in turn affect
the 260 nm reading for DNA and RNA. A number of substances absorb at 230 nm, as this is
the region of absorbance of peptide bonds and aromatic side chains. Several buffer
components exhibit strong absorption at 260 nm and therefore can alter the results of
photometric quantification. One example of such a component is EDTA in concentrations
above 10 mM. Contaminants in a sample, such as proteins, phenol, or urea, can result in
absorption at 230 nm. Phenol contamination also increases a sample’s absorption at 280 nm
and therefore can be identified through a lower A260/A280 ratio. An A260/A230 ratio of 2 or above
is indicative of a pure sample.

A 320 Background correction

Background correction is a process whereby the absorption at a point on the spectrum unrelated
to the sample being analyzed is also measured, and the reading subtracted from the peaks.
Absorption at 320 nm may be due to light scatter caused by particles, or to a precipitate in the
sample. Dirty or damaged cuvettes can cause absorption at 320 nm. Contaminations with
chaotropic salts, such as NaI, can also lead to increased light scatter.

Measuring and correcting for the reading at 320 nm therefore removes any interference from
light scatter, from the cuvette, or in cases where a blanking plate is used to target the light beam
though the sample.

Background correction is particularly useful when using small volume cells or specialist small
volume spectrophotometers.

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Nucleic acid measurements with low volume instruments
To get meaningful results, as a rule of thumb, the following two criteria must be met:

• Abs260 > approx. twice Abs230 (A260/A230 Ratio=2)

• Abs260 = 0.1 or more (indicative of a high enough concentration, ie solution not too dilute)

Typically the only measurements checked for DNA measurement are concentration and
A260/A280 ratios. Unexpected results can often be explained by looking at the underlying A230,
A260, A280, A320 values.

As an example:

Sample 1 Sample 3
Concentration 10.7 µg/ml Concentration 11.0 µg/ml
A230 A260 A280 A320 A230 A260 A280 A320
9.27 0.244 0.129 0.030 9.32 0.326 0.211 0.107

A260/A280 A260/A230 A260/A280 A260/A230

2.162 0.023 2.106 0.024

Sample 2 Sample 4
Concentration 11.1 µg/ml Concentration 10.5 µg/ml
A230 A260 A280 A320 A230 A260 A280 A320
9.33 0.323 0.206 0.101 9.30 0.303 0.192 0.094

A260/A280 A260/A230 A260/A280 A260/A230

2.114 0.024 2.133 0.023

In this example, the instrument is giving concentration values which are very close together
(<2% variation). So the reproducibility for concentration values is within the expected values.
However, looking at the underlying absorbance readings shows that the 230 nm reading is very
high compared to other readings making the ratio A 260 /A 230 very low. A value < 1.8 indicates
potential contamination.

Note that in this example

• The background at A320 nm is provided for information & must be subtracted from
the A260 nm reading to arrive at the absorbance
• For information: Not all low volume instruments display the A 320 nm subtraction in
the calculation.

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Protein applications
As with DNA, proteins absorb light at a specific wavelength, allowing direct measurement using
a spectrophotometer. The amino acids Tyrosine and Tryptophan have a very specific absorption
at 280 nm, allowing direct A280 measurement of protein concentration. Direct UV measurement
at 280 nm has many advantages, since the protein solution alone is used, without the addition
of reagents, and it is not modified or inactivated during the process. No incubation period is
required, so measurements are quick and highly reproducible.

The chemical composition of the protein will affect the absorption: the number as well as the
type of amino acids will cause variation. How much a protein absorbs at 280 nm is dependent
on the amount of the amino acids Tyrosine and especially Tryptophan: the aromatic ring of
Phenylalanine absorbs well at 260 nm, but not 280 nm. So proteins of similar molecular weight
can have quite different absorbances, since they can have completely different Tryptophan and
Tyrosine content. UV absorbance of aromatic side chains is also affected by protein structure.
Therefore conditions which affect structure, such as temperature, pH, ionic strength, or the
presence of detergents, can affect the ability of aromatic residues to absorb light at 280 nm,
and change the value of the protein’s extinction coefficient.

As with nucleic acids each protein has its own conversion factor. The common standard protein
bovine serum albumin (BSA) has a factor of 1.551.

Concentration (µg/ml) = Abs280 × Factor

The A260/A280 ratio can be used as a guide to the purity of the sample.

Some instruments also contain factors for other common proteins such as BSA or IgG which
allow users to choose the protein closest in type to their sample, if the factor for the sample
of protein is unknown.
2.2
X: 300
2.0
Y: 0.049
1.8
1.6
1.4
Absorbance

1.2
1.0
0.8
0.6
0.4
0.2 ×
0.0
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)

Fig 5. A typical wavelength scan for a protein sample.

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Protein concentration calculations
The protein concentration, c, in mg/ml is calculated by:

c = A280/(E280,1 mg/ml) × l)

The absorbance coefficient (E280,1 mg/ml) corresponds to the A280 of a 1 mg/ml solution of the protein
and varies between proteins.

E280,1 mg/ml can be determined:

1. by measuring the absorbance of the protein in a solution of known concentration; or

2. by the theoretical calculation

E280, 1 mg/ml = (5500nTrp + 1490nTyr + 125nS-S)/M

where nTrp, nTyr and nS-S are the number of Trp and Tyr residues, nS-S is the number of disulfide
bonds (S-S bonds) in the protein sequence, and M is the molecular weight of the protein.
Coenzymes and cofactors may also contribute. Examples of values for E280, 1 mg/ml include
0.67 for BSA, 1.37 for IgG, and 2.64 for lysozyme.

Light scattering correction of the A280 value can be made by:

A280 = A280 (measured) – 1.929 × A330 (measured)

Christian and Warburg (3)

Nucleic acids have absorbance at 280 nm (maximum at 260 nm). If the presence of nucleic acids
is suspected, the protein concentration can be estimated (with less accuracy) according to
Christian, W. and Warburg, O. (3):

C (mg/ml) = 1.55 × A280 - 0.76 × A260

The constants 1.55 and 0.76 refer to a specific protein used by Christian and Warburg. For best
accuracy, the factors should be determined for the target protein at hand. Refer to the
NanoVue™ Plus User Manual, 28-9574-75 from GE Healthcare.

Colorimetric methods
A wide variety of colorimetic methods for protein concentration measurements are available,
they all work in a similar way.

The reagents contain a dye which binds specifically to the proteins in a solution. The absorption of
a known set of standards is measured and a standard curve produced. Absorption measurements
of unknown samples can then be compared to the curve to establish their concentration.

It is important to check the working ranges of the commercial product being used, as these
can vary.

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BCA (4)
This method relies on the reaction of cupric ions (Cu2+) and peptide bonds. This forms Cuprous
ions (Cu+) which are then detected with bicinchoninic acid (BCA). This gives an absorbance peak
maximum at 562 nm. Most commercially available kits state that this method is designed to
quantify 125 to 2000 µg/ml.

Biuret (5)
Like BCA, the Biuret method takes advantage of the reaction between cupric ions and peptide
bonds in alkali solution. In this case no additional development step is used. Absorption is
measured at 546 nm. Most commercially available kits state that this method is designed for
the quantification of 1000 to 15 000 µg/ml.

Bradford (6)
This method uses a dye, Coomassie Brilliant Blue, which binds to the protein. The protein
solution shows an increase in absorbance at 595 nm which is proportional to the amount of
bound dye. Bradford is very resistant to interference making it a very common method. Most
commercially available kits state that the useful range of this method is 1 to 1500 µg/ml.

Lowry (7)
Lowry takes advantage of the reaction of Folin-Ciocalteu’s phenol reagent with tyrosyl residues
of an unknown protein. The absorption at 750 nm can then be compared to a standard curve
of a known protein, normally BSA. Most commercially available kits state that this method is
useful to quantify 1 to 1500 µg/ml.

2-D Quant Kit

The 2-D Quant Kit (80-6483-56) is designed to determine protein concentration in samples
to be analyzed by high resolution electrophoresis techniques such as 2-D electrophoresis,
SDS-PAGE or IEF. Many reagents used in preparing such samples, including detergents,
reductants, chaotropes and carrier ampholytes, are incompatible with other protein assays.
The procedure quantitatively precipitates proteins, leaving interfering substances in solution.
The assay is based on the specific binding of copper ions to protein. Precipitated proteins
are resuspended in a copper-containing solution and unbound copper is measured with a
colorimetric agent. The color density is inversely related to the protein concentration. The assay
is linear in the range of 0–50 µg protein. The procedure is compatible with 2% SDS, 1% DTT,
8 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte™ and 2% IPG Buffer. Absorbance is read
at 480 nm.

See the following pages for a summary you can use for choosing your protein assay method.

Please refer to the kit manufacturer’s product data sheets for the exact ranges for your
specific kit.

11
Glossary
Absorption
Absorption is the amount of light that a substance takes in and does not allow to pass through
it. Spectrophotometers actually measure transmission, the amount of light that passes
through a sample, but this is converted into absorption by comparing the bulb output to the
light that has passed through the sample.

Bandwidth
Bandwidth describes the property of the light emitted from the monochromator. When the
light is analyzed, the peak may be at its maximum at the specified wavelength, but light is also
emitted to a lesser extent at the wavelengths either side of the one selected. The Spectral
Bandwidth is a measurement of the width of a peak at half-light intensity (Fig 9).
Intensity

Half Height

Spectral
Bandwidth

Wavelength
Fig 9. The Bandwidth is a measurement of the width of a peak at half-light intensity.

To obtain maximum sensitivity, the bandwidth of the instrument would cover the entire
bandwidth of the substance being measured. When reducing bandwidth, the amount of
available light for absorption is also reduced, so more sensitive detectors are needed to
obtain the same working range.

Increasing bandwidth reduces resolution, and measurement at too broad a bandwidth could also
possibly merge two absorption peaks into one larger one, giving an artificially high reading.

Low bandwidth instruments are very important in highly regulated environments such as
pharmaceutical and Pharmacopoeia laboratories where resolution of 1 nm is often required,
whereas a broader bandwidth is helpful for sensitive DNA quantification where absorption is
strong, and only one, broad, 5 nm peak is being measured.

24
Quantitative analysis
A single wavelength measurement of a number of known standards can be taken, from which
the instrument creates a standard curve, to which future measurements of unknown samples
can be compared to establish their concentration.

Cell Density
Light scatter from bacterial cell cultures can be used as an indication of cell concentration and
when plotted over time can be used to see if the culture is ready to be harvested. Measurements
are taken at 600 nm.

Monochromator
In a UV/Vis spectrophotometer, a monochromator is the optical device that transmits a
mechanically selected band of wavelengths of light from a wider range of wavelengths
available from the light source. From the Greek mono (single) and chroma (color).

Pathlength
Pathlength is the distance through the sample, through which the light has to pass, in order to
reach the detector. Standard cuvettes have a pathlength of 10 mm (1 cm). GE Healthcare offers
instruments covering pathlengths from 0.2 mm all the way to 100 mm.

Stray Light
Stray light is light that reaches the detector that is of a different wavelength to that the
monochromator is selecting. This can be caused by the diffraction pattern produced by the
monochromator or from light leaks from the outside environment. GE Healthcare systems
are designed to reduce all possible sources of stray light.

UV/Vis
The electromagnetic spectrum is split into a number of different regions by wavelength. These
include X-ray (0.01-10 nm) Visible light (380-760 nm) and Microwaves (1-10cm). GE Healthcare
UV/Vis spectrophotometers all measure in the UV (10-400 nm) visible (380-760 nm) and near
infra-red (750-2250 nm) regions covering a range of 190-1100 nm.

Wavelength
Represented by the lower case Greek letter lambda λ. Wavelength is a measurement of the
distance between successive peaks in a waveform.

Wavelength
λ

Distance
27